Electrochemical Sensing Technology For Detection Of Enteropathogenic Bacteria

The electrochemical sensor can sense the changes of physical andchemical which was directly or indirectly caused by analytes, and convert itinto an electrical signal to analyze the analytes. In recent years, there is greatprogress in the electrochemical sensor research by integrating microbiology,molecular biology, analytical chemistry, materials science. It can detect alarge number of substances rapidly and accurately, including proteins,nucleic acids, inorganic ions, drugs, microorganisms, etc, and it is a veryactive research in the laboratory medicine and analytical science. In ourwork, a rapid、simple、sensitive and specific method for detection ofenteropathogenic bacteria based on electrochemical sensor was developedsuccessfully, which would become a powerful tool for clinical diagnostics,food safety and environmental monitoring.These contents include thefollowing three parts:1. A rapid and sensitive aptamer-based electrochemical biosensor fordirect detection Enteropathogenic Escherichia coliA sensitive and specific electrochemical biosensor based ontarget-induced aptamer displacement was developed for direct detection of EPEC. The aptamer for EPEC was immobilized on a gold electrode byhybridization with the capture probe anchored on the electrode surfacethrough Au-thiol binding. In the presence of EPEC, the aptamer wasdissociated from the capture probe-aptamer duplex due to the strongerinteraction between the aptamer and the EPEC. The consequentsingle-strand capture probe could be hybridized with biotinylated detectionprobe and tagged with streptavidin-alkaline phosphatase, producingsensitive enzyme-catalyzed electrochemical response to EPEC. Under theoptimal conditions, the established strategy could directly detect EPEC withthe detection limit of112CFU mL-1in phosphate buffer saline and305CFUmL-1in milk within3.5h, demonstrated the sensitive and accuratequantification of target pathogenic bacteria. The designed biosensor couldbecome a powerful tool for pathogenic microorganisms screening in clinicaldiagnostics, food safety, biothreat detection and environmental monitoring.2. A sensitive electrochemical DNA biosensor for specific detection ofEnterobacteriaceae bacteria by Exonuclease III-assisted signal amplificationA specific and sensitive methodology was developed successfully forquantitative detection of Enterobacteriaceae bacteria by integratingExonuclease III-assisted target recycling amplification with a simpleelectrochemical DNA biosensor. After target DNA hybridizes with captureDNA, Exonuclease III (Exo III) can selectively digest the capture DNA,which releases the target to undergo a new hybridization and cleavage cycle on sensor surface, leading to a successful target recycling. Finally, the leftcapture DNA is recognized by detection probe to produce the detectablesignal, which decreases with the increasing target DNA concentration.Under the optimal conditions, the proposed strategy could detect targetDNA down to8.7fM with a linear range from0.01pM to1nM, showinghigh sensitivity. Meanwhile, the sensing strategy had successfully been usedfor detection of Enterobacteriaceae bacteria down to40CFU mL-1in milksamples. This strategy presented a simple, rapid and sensitive platform forEnterobacteriaceae bacteria detection and would become a versatile andpowerful tool for food safety, biothreat detection and environmentalmonitoring.3. A sensitive and simple strategy for Enteropathogenic Escherichiacoli detection using a bfpA gene-based electrochemical DNA sensorA sensitive and simple strategy for the detection of EnteropathogenicEscherichia coli (EPEC) was developed by integrating simple DNAextraction, specific polymerase chain reaction (PCR) with a bfpA gene-basedelectrochemical DNA sensor. The PCR amplified products with bfpA genecould be specifically captured on the electrode, and further hybridized withbiotinylated detection probes to form a sandwich hybridization structurewith two biotinylated detection probes. Then the sandwich hybridizationstructure with two biotinylated detection probes significantly facilitated thenumerous streptavidin alkaline phosphatase (ST-AP) on the electrode, and enzymatic electrochemical readout for dual signal amplification.The developed electrochemical DNA sensor could discriminatesatisfactorily the complementary and mismatched oligonucleotides,showing high specificity. Under optimal conditions, this strategy showed ahigh sensitivity for synthetic target DNA in a concentration range of0.001-10nM with a detection limit of0.3pM. Meanwhile, the novel DNAsensor could quantitatively detect EPEC from10to107CFU mL-1within3.5h. This electrochemical DNA sensor provided a pragmatic platform forconvenient detection of EPEC and would become a powerful tool forpathogenic microorganisms screening in clinical diagnostics, food safetyand environmental monitoring.